Ribonucleotide reductase (RR) key to maintaining life as it catalyzes the slowest step in de novo DNTP synthesis by reducing ribonucleotides to deoxy ribonucleotides. RR is a multi-subunit enzyme consisting of a subunit that contains two allosteric sites and a catalytic site, and a b subunit that houses a free-radical that initiates thol-based catalysis. For almost 40 years RRs were thought to exist as heterotetramers. This organization failed, however, to explain how dATP inactivates and ATP activates the enzyme. Recently, this view has been challenged by biochemical data from other labs and structural data reported by us that support the existence of higher-order oligomers induced by its activator ATP and its inhibitor dATP. Interestingly, though ATP is an activator and dATP is an inhibitor of RR, they both cause the subunit to hexamerize. We propose that the two hexamers have different packing arrangements which lead to opposite outcomes of RR activity. Additionally, two important cancer drugs, gemcitabine and clofarabine are known to bind to higher-order RR oligomers. Thus, it is becoming increasingly clear that for an understanding of how RR is regulated in eukaryotes and targeted by cancer drugs, it is essential to elucidate the structure of higher-order oligomers formed by eukaryotic RRs. As higher-order holo-complexes may not be amenable to X-ray crystallography, we propose to use X-ray crystallography to determine the high-resolution structures of RR1 oligomers and single-particle electron microscopy (EM) to elucidate the organization of RR1 and RR2 in the holo-complexes. PUBLIC HEALTH RELEVANCE: Ribonucleotide reductase (RRs) is a multi-subunit enzyme consisting of a large and small subunit. Recent studies show that the allosteric activator ATP and inhibitor dATP regulate RR by inducing hexamerization of the large subunit. This is a departure from the previously held view of a RR heterotetramer. In the current proposal, we will investigate how RR is regulated by ATP and dATP through oligomerization and how RR oligomers are targeted by cancer drugs using X-ray crystallography, cryo-EM and site- directed mutagenesis.